It is a central idea of the NIH that basic research will lead to new approaches in medicine, and we believe that we have found one. As a result of earlier funding by the NIH, we have discovered a family of peptides that (1) target acidic tissues in vivo, including tumors, and (2) can deliver polar molecules into cells, releasing them in the cytoplasm. Studies of the peptides have led to new insights about the basic science of peptide insertion into membranes in terms of structure, energetics and kinetics, and as we learn more about the principles, we will be able to design better versions of peptides for specific applications. The peptides, which we call pHLIPs (for pH (Low) Insertion Peptides) are soluble as mostly unstructured monomers in aqueous solution, bind as unstructured monomers to the surfaces of bilayers or membranes, and fold to make helices that insert across membranes when the environment is acidic. We have established the basic energetics and kinetics of peptide insertion. We have shown that a labeled version of pHLIP targets and images many kinds of tumors in mouse models, and that the imaging reveals very small tumors (1mm) and accurately identifies tumor borders. We have also established that small cargo molecules can be delivered into cancer cells on the inserting end of pHLIP. A water-soluble therapeutic molecule can be attached as cargo to the inserting end of pHLIP by a bond that is unstable in side a cell, but stable outside the cell. When pHLIP folds at low pH and delivers the cargo across the membrane, the bond breaks and the cargo is released in the cytosol, where it may have a therapeutic effect. When injected into a mouse, a form of pHLIP-delivered treatment has been recently demonstrated to successfully target and effectively treat lymphoma in a mouse model. Our proposed studies are aimed at refining pHLIP designs for specific uses in targeted delivery of toxins and Peptide Nucleic Acids. We also plan a more advanced method for fluorescence labeling of tumors and a new method for measuring the pH at cell surfaces, a key parameter for the acidic targeting of pHLIPs. If successful, while learning more about the biophysics of peptide-membrane interactions and the physiology of cell surface acidity, we will have advanced the technology toward clinical applications.